Dysregulation of androgen receptor (AR) signaling plays a critical role in different phases of progression of prostate cancer (PCa). The AR-mediated anti-apoptosis pathway is enhanced in PCa, which is accompanied by inhibition of AR-mediated pro-apoptosis pathway such as down-regulation of a tumor suppressor gene U19/Eaf2. Thus restoration of AR signaling may represent an effective approach for the treatment of PCa. In this application we propose to develop a novel strategy to synergistically inhibit AR signaling in hormone refractory PCa cells via co-delivery of AR specific siRNA and U19/Eaf2 transgene. Delivery of the combined therapeutics will be achieved via a new polymer that provides enhanced transfection and significantly decreased toxicity. To ensure sufficient stability in blood circulation to allow sufficient time for the nanoparticles to home to PCa, these nanovectors will be surface-shielded with a molecule that is programmed to de-shield once the particles are exposed to a weakly acidic environment of endosome. To achieve specific targeting the vector will be further decorated with a small molecule ligand for PSMA. Three specific aims will be pursued. Aim 1: To achieve efficient co-delivery of AR siRNA and U19/Eaf2 transgene to PCa cells using a smart polymer-based, PCa-specific nanovector. Cell type-specific delivery of fluorescence-labeled siRNA or U19/Eaf2 will be examined with PSMA-positive and PSMA-negative PCa cell lines and the efficiency will be compared to that of ligand-free nanoparticles. Targeted delivery of siRNA/U19/Eaf2 will be then examined in tumor-bearing mice. Aim 2: To investigate the therapeutic effect of co-delivery of AR siRNA and U19/Eaf2 expression plasmid in cultured PCa cells. The biological consequence of targeted delivery of AR siRNA/U19/Eaf2 will be investigated by examining: a) changes in the expression levels of AR and its target gene, PSA (via qRT-PCR, Western, and ELISA), and b) cell growth (via MTT and staining of apoptotic cells). Controls of scrambled siRNA and mutated U19/Eaf2 will be included to elucidate sequence-specific effect. Aim 3: To conduct preliminary studies to investigate the therapeutic effect of co-delivery of AR siRNA and U19/Eaf2 expression plasmid in an animal model of PCa. The in vivo therapeutic effect of targeted delivery will be addressed in an s.c. animal model of human PCa. In addition to monitoring the tumor growth, we will examine the expression levels of AR and PSA at the completion of the studies. Successful completion of this study may lead to the development of a novel therapy to advance the treatment of prostate cancer. PUBLIC HEALTH RELEVANCE: This study is to develop a combined therapeutics that is synergistically targeted at the androgen receptor signaling in prostate cancer. Successful completion of this study may lead to the development of a novel therapy for the management of advanced prostate cancer.